Blood test composition and method



BLOOD TEST COMPGSITIQN AND METHOD Richard S. Nicholls and Dale E. Fonner, Elkhart, Ind., assignors to Miles Laboratories, Inc, Ellrhart, Ind, a corporation of Indiana No Drawing. Application April 15, 1954, Serial No. 423,524

Claims. (Cl. 252-408) This invention which relates to a diagnostic composition for detecting occult blood in animal fluids is a conatnt The present invention is directed to a tablet which is 7,

adapted for use in determining occult blood in such materials as urine and the like or in fluids containing urine which tablet is superior to the tableted compositions now available in several important respects, namely, in that with the present tablet the time of reaction is much more I uniform for any given concentration of blood, and fuf- I thermore, the sensitivity of the tablet within a given time is substantially and unexpectedly greater.

Generally, the tablet of our invention is formed of a' mixture of a blood indicator such as, for example, o-

tolidine, o-toluidine, benzidine, o-tolidine-dihydrochloride and similar compounds; an oxidizing agent of the peroxide type such as sodium carbonate peroxide, sodium perbor silicate, strontium peroxide, sodium pyrophosphate peroxide, barium peroxide, magnesium peroxide, and urea peroxide; an acetate such as calcium acetate, sodium acetate, potassium acetate, lithium acetate and the like; a water insoluble pigment or dye; and a water-soluble solid acid which is stronger than acetic acid so that it can react with the aforementioned acetate salt to produce acetic acid. Examples of such acids are citric, fumaric, itaconic, maleic, rnalic, malonic, and mandelic acids. Additionally, there must be present an effervescent couple comprising, for example, an acid in solid dry form and a carbonate or bicarbonate which is preferably any alkaline earth or alkali metal carbonate or bicarbonate such as sodium, potassium, and calcium carbonates and bicarbonates. Diluents and excipients' of the kind conventionally used in preparation of tablets are likewise used in conjunction with the aforementioned materials.

The above composition is prepared in tablet form, each tablet containing conveniently 2 grains of material and being conveniently A; inch thick, and about A inch in diameter. In determining the presence or absence of occult blood in a sample of urine a drop of the urine specimen is placed on a piece of dry filter paper, and when the drop has soaked into the paper a tablet prepared as above described is placed in the center of the drop and then two drops of water added to the tablet. With a positive test a ring of color appears on the filter paper surrounding the tablet, the color ranging fiom a very faint to a very deep blue, depending upon the concentration of blood in the sample. The higher the blood concentration, the deeper the color, and vice versa. The afore- 2,799,660 Patented July 16, 1957 said method of determining occult blood is found superior to the methods taught by the prior art, not only in the degree of sensitivity reached but also in the greater extent of reproducibility.

In connection with the composition and method of the present invention, it is absolutely essential that the composition contain, in addition to the blood indicator and an oxidizing agent, an acetate, a water soluble organic acid such as tartaric acid which is capable of releasing acetic acid when contacted with an acetate, and an effervescent couple. Without the co-presence of these materials neither the degree of sensitivity nor the degree of reproducibility of test results will be adequate.

While the formulation of our diagnositic composition may be varied in accordance with the procedures known to those skilled in the art, suitable formulations are as follows:

Strontium peroxide From '5 to 15, preferably 9. Tartaric acid From 20 to 60, preferably 30. Calcium acetate From 20 to 75, preferably 60.

Corn starch From 1 to 10, preferably 5. Effervescent couple From 5 to 20, preferably 10.

EXAMPLE II Components of reagent mixture:

o-Tolidine From 0.5-4, preferably 1. Stront um peroxide From 5-15 preferably 7.5. Succ mc acid From 20-75, preferably 40. Calcium acetate From 20-75, preferably 60. Corn starch From 1-10, preferably 5. Effervescent couple From 5-20, preferably 10.

EXAMPLE HI Components of reagent mixture:

o-Tolidine From 1-6, preferably 4. Sodium perbor silicate From 8-20, preferably 10. Succrnic acid From 20-75, preferably 40. Calcium acetate From 20-75, preferably 60. Corn starch From 1-10, preferably 5. Effervescent couple From 5-20, preferably 10.

EXAMPLE IV Components of reagent mixture:

o-Tolidine From 0.5-4, preferably 1.

Strontium peroxide Itaconic acld Calcium acetate Corn starch Effervescent coup From 5-15, preferably 7.5. From 20-75, preferably 40. From 20-75, preferably 60. From 1-10, preferably 5.

. From 5-20, preferably 10.

EXAMPLE V Components of reagent mixture:

Benzidine hydrochloride From Strontium peroxide 'lartaric acid 1-5, preferably 1. 5-15, preferably 7.5. 20-60, preferably 30.

Calcium acetate From 20-75, preferably 60.

Corn starch From 1-10, preferably 5.

Efiervescent couple From 5-20, preferably 10.

EXAMPLE VI Components of reagent mixture:

o-Tolidine-dihydrochloride- From 1-5, preferably 1.

The present invention is particularly useful in the determination of occult blood in the presence of urine and, in sharp contrast with diagnostic compositions of the prior art, is sensitive to the extent of detecting of one part blod in 100,000 of uncentrifuged urine.

The net effect of the effervescent couple and the acetate components of our formulation is to increase substantially the sensitivity, reproducibility and the speed of the test procedure. This is illustrated by tests performed with the following four (4) compositions, each being prepared as a 2 grain tablet:

4 only in trace amounts sufficient to establish a color tint in the tablet. Thus for the water insoluble coloring material D and C Red No. 35 it has been found that approximately one part in 1,000 parts of the remaining Tablet A 5 composition is adequate. Other suitable colors are D parts by Weight and C Yellow No. l1 and D and C Orange No. 17. HowoJIofidi 1 v r, almost any insoluble pigment or dye which con- Strontium peroxide 9 trasts strongly with blue would be satisfactory unless it Tartaric acid 30 sho ld In some other way interfere with the test as by Starch 5 10 reacting with another ingredient of the tablet. It is to Talcum 5 the general formulations of Examples I to VII above as 7 represented by tablets C and D that this application is Tablet B related, the peculiar merits of such formulations being Parts by welght made especially evident by comparison with the perf 1 formance of the other compositions of tablets A and B. strontium .Peroxlde 9 These tablets were tested as follows: 'f 30 Blood was diluted with normal urine to prepare sam- Calcmm acetate ples which contained blood in concentrations of 1:100,000, starch 5 l:20,000, and 1:40,000 parts of urine. To make the Talcum 5 test one drop of the specimen was placed on a filter aper T bl t C square laid out on a white tile. When the drop had Pa t by weight soaked into the paper a tablet was placed in the center o-Tolidine 1 of the wetted area and then two drops of Water added Strontium peroxide 9 so that they fell on the tablet and ran over onto the Tartari acid 30 paper. The examination of the test was made at the Calcium acetate 60 end of 2, 3, 4 and 5 minutes, the time being determined Starch 5 by a stop watch. Talcum 5 Each type table was tested a large number of times Efiervescent couple (tartaric acid and sodium biwith each of the 3 concentrations of blood. The results carbonate) 10 are recorded in Table I.

TABLE I Tablet A Tablet B Tablet 0 Concentration of blood added to urine Percent positive in Percent positive in Percent positive in No. No. No. Tests Tests Tests 2 3 4 a 2 3 4 5 2 3 4 5 min. min. min. min. min. min. min. min. min. min. min. min.

Tablet D Parts by weight o-Tolidine 1 Strontium peroxide 9 Tartaric acid 30 Calcium acetate Starch 5 Talcum 5 Effervescent couple 10 Water-insoluble pigment or dye 0.1

It will be noticed that tablet A is of the general type of prior art compositions such as are described, for example, in U. S. Patent 2,290,436, that tablet B is the composition of tablet A to which calcium acetate has been added and that tablet 'C is of the same composition as tablet A to which both calcium acetate and efliervescent couple have been added.

Tablet D is the same composition as tablet C so modified as to include a water-insoluble pigment or dye. To incorporate the pigment or dye in the tablet it is simply necessary that it is dry and finely divided and it is then mixed intimately with the remainder of the composition prior to compressing the material into tablets. The amount of pigment required would obviously vary depending on its hiding power and opacity, therefore the amount considered most satisfactory for any given pigment must be determined by trial. However, the necessary amount is generally not very much, being present The superior performance of tablet B over tablet A shows that the addition of calcium acetate increases considerably'the sensitivity of the test. Further it is obvious that tablet C is more uniform in reaction time than tablet B and in addition the sensitivity of the tablet within a given time period is very substantially improved.

In the practice of our invention we have found that our test procedure which employs filter paper provides a much more sensitive test than other methods such as placing a drop of the sample directly on the tablet or dissolving the reagent mixture in water and adding it to the specimen. Under these latter conditions tablet A gives a negative test for blood at 1:0,000 and indeed only a very slowly formed positive with 1 part of blood in 1,000 parts of urine. The sensitivity of any of the compositions to blood dissolved in water is much higher than it is to blood in urine. Urine appears to contain a factor which inhibits the formation of color with o-tolidine, benzidine, etc., in the presence of blood and a peroxide. The use of the filter paper seems to a large extent to get around this difliculty and brings about a marked increase in sensitivity.

The tablets B and C were both compared by testing them with the sediments obtained by centrifuging samples of urine containing very small quantities of blood. In this case blood was diluted with normal urine to produce blood concentrations of 1 part in 50,000; 1 part in 100,- 000; 1 part in 300,000 and 1 part in 1,000,000 parts of urine. In making the test 15 cc. of one of the specimens was centrifuged in a conical tube at moderate speed. The *the mannerdescribed can be illustrated by the following supernatant liquid was drawn off and the sediment which remained tested by placing 1 drop of it on filter paper and then proceeding with the test as previously described.

record. j V

A number of urines wereobtained which were judged by microscopic examination of the sediment to contain The results of a series of tests are recorded in the follow- 5 no red blood cells, and could thereforebe expected to. ing table. give negative tests, that is there should be no blue color- TABLE II [Tests on sediment after centrifuging] Tablet B Tablet Concentration of blood added to Percent positive in Percent positive inunne No. No.

Tests 2 3 4 5 Tests 2 8 4 5 min. min. min. min. min. min. min. min.

It is again seen from the above results that with tablet ng on the P p Surrounding the tablet material- A C positive readings were obtained more consistently and Series f SW carried Out in Which a number of reliably than when tablet B was used. operators tested the two tablet compositions witheach Notwithstanding the very meritorious results obtained 0f the mines but in Such a manner that y did not by the use f t bl t C as d ib d b there i some run the tests with the two tablets concurrently and so were slight diificulty in deciding whether the test employing unable to compare their findings with one composition tablet C reads positive or negative when there is extremely against their findings With another on the Same urine little or no blood in the test sample. In these situations P Altogether 600 tests were carried out with each the subjective viewpoint of the person reading the result eempeslheh, the readings being taken at theme of 2 has considerable effect since he is not guided by any clearly mlhuteswith tablet C, the testshh these negative mines d fi d 1 h were considered to show at least. a faintly positive test When using such a tablet as represented by tablet C m 7 cases 125% of h tests run, he in h in the manner described above, the test is considered to 35 .Senes of teste Performed Wlth T eempeslheh e be positive when blue color appears on the paper surmg a red Plgment, tablet m the manner desenbed rounding the reacting tablet. However, it is a fact that above q 9 taste or 15% of the total e eonsldered as the effervescent tablet reacts, the material of the as jhowmg anythmg but a clear negahve test; tablet sloughs oif onto the paper in the area immediately lhe tablet of the Present mventlon thus Provides an around the tablet. In places where the amount on the 40 lmhmved mhans for detechng fields Such as paper is relatively little it is sometimes difficult to decide h by which Ihhhhs vhthal ehhhhahoh of false whether the paper should be considered as having turned poslhve readings 1S achleved 1h h dhhchhy detectable blue or whether the blue color which can be seen is areas of ehremely. 10W cohcehtrahoh complete h some spilled over tablet material since there is a tendsehce h hh Wlth reshlhhg marked Improvement ency for the test material to take on a little gray or very test rehhhhlty pale blue color. Further than this, even when there is Havlhg thus described 9 lhvhhhoh 9 no question as to which is material and which is clear A hloohfdetechhg dlhghoshc composmoh m tablet paper there can be confusion from the shadows which form. hhhpnslhg a.c.ompohhd hh h the group the tablet throws on the paper because these often have h i of h ohohhdihe hehzldlhe h a light grayish cast and are irregular in outline since the tohdme dlhydrohhlonde a peroxlde which liberate tablet on efiervescing has lost its usual outline. hydrogehherohlde a Water Soluble .so'hd .ahld Stmhger We have found that these diiliculties can be eliminated than ahehc h acetate salt whlch react Wlth and the use of the test very greatly improved by the dethe Sald Sohd held to phohhce achhc h ah ehhervhh vice of coloring the material of the test tablet with a h and a Watehlhsolhhle hihterhh cohtrashhg coloring material which contrasts with the blue color of In Color h the color formed by sald color responslve a positive test in such a way that the contrasting color compouhh In Presehcehf i remains with the reagent material and does not diffuse A dlaghoshc composlhoh h tablet fohhhomphslhg out on its own into the paper and so hide the appearance ahlember h the group hh of h of a possible positive reaction. To achieve these objects h behzhhhe h .o'hhuhhhe dlhydrochlohhe a Perthe added coloring material should be a water-insoluble Ohlde Whlhh h hberate hydrogeh. pehoxlde Water pigment or dye so that it will not leach away from the soluble .sohd.acld strohghr than achhc i ah acetate reagent material with which it is combined in the tablet Shh h whl rhact Wlth the last sand and to Produce when the whole mass is wetted as described in the test. held ah ehervhsceht h and a water'soluble In addition, in order that the coloring material should perh h cohtrashhg m color Wlth the 00101 formed form its function of defining clearly where the reagent m combmatloh Wlth bleed by the Selected member of material is at the completion of the test, it is important Sald groupthat its color contrast strongly with the blue color which A diagnostic composition in a l t form for deforms on the paper when the test is positive and for this tecting blood in the presence of urine comprising by reason yellow, orange or red pigments are the most satisg from t0 4 P3115 O'tOIidhIle, 5 to 15 P511rts factory. Further, the coloring materials should have the strontium peroxide, 20 to 60 parts tartaric acid, 20 to property of good opacity and hiding power so that they parts calcium acetate, 1 to 10 parts corn starch, 5 to 20 shall stand out well in contrast to blue and further they parts of a solid effervescent couple, and a minor amount shall cover up any tendency of the material of the test of a water-insoluble material contrasting in color with itself to turn blue. 75 the color formed in combination with blood by said The advantage of coloring the tableted composition in o-tolidine.

8.--A diagnostic. composition-in tablet form comprisinglarmemberaofthe-group consisting-of o-tolidine, o--

toluidine. benzidine, o-tolidine ldihydrochloride, a, peroxidefor liberating hydrogen peroxide, alwater soluble 5 solidjacid stronger. than acetic acid and an effervescent couple, an acetic" sa'lt'which will react with the said solid acid to produce acetic acid, said tablet efiecting a color change on urine Wetted filter paper when placed thereon, and said tablet is contacted with asmall amount of form comprising a compound selected from thettgroupJfl W enwhen Said urine containslblood.

consisting of o-tolidine, o-toluidine, benzidine and o-tolidine dihydrochloride, a peroxide which will liberatelhy: drogen peroxide, an acetate selected from the group;-

consisting of calcium acetate and alkali metal acetates 9. A method of determining occult blood in urine which comprises rwettingma portion of dry filter paper withaurine, placing-ontthe ..urine wetted portion of said paper, a tablet, composed of a mixture comprising by a water soluble acid stronger than acetic acid, and Ian 15 gh fIOm-abOI-It Parts of wmpound effervescent couple.

6. A diagnostic composition in tablet form f0'l' 'dfi' tecting blood in the presence of urine comprising by weight from 0.5 to 4 parts o-tolidine, 5 to parts of 75 parts of calcium acetate, 1 to 10 parts cornstarch, and 5 to parts of a solid eflervescent couple, said tablet effecting a color change on urine wetted filter paper when placed on'suchlpaper andthe 'tablet'conta-cted with a small amount of water.

7. A'diagnosti'c 'composition'in tablet form for detecting blood intliepresence of urine comprising 1 part of 'o-tolidine; 9parts of strontium peroxide, parts of tartaric acid, parts of calcium acetate, 5 parts of 'cornstarch, and 10'parts' of'a solid efiervescent couple, the 30 tablet efie'ctingea color change on urine-Wetted filter paper upon which said "tablet is placed, after a small amount of water is placed on said 'table't.

lected from the-ugroup'consistingof o-tolidine, o-toluidine; benzidine' and o-tolidine dihydro-chloride; a peroxidezwhich liberates hydrogen peroxide, an acetate se-- lected from the group consisting of calcium acetate and strontium peroxide, 20 to 60 parts of tartaric acid, 20 to 20 alkali metal acetates, about 20 to Parts by Weight a water solublesolid acid stronger than acetic acid, and' about 5 to 20 parts of an eifervescentcouple and then adding'a small amount of water to said tablet.

10. The composition of claim 2 wherein the said water- 5 insoluble material is'selected from the group consisting of red pigments, yellow pigments, and orange pigments.

Kamlet July 21, 1942 Compton et al Oct. 23, 1945 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 2,799,660 July 16, 1957 Richard S, Nicholle et 81.

It is hereby certified that error appears in the printed specification of the above numbered patent requiring correction and that the said Let oers Patent should read as corrected below.

Column 6, line 60, before "water" insert a line 63, after "acetic" strike out the comma; same line, for "water-soluble" read water-insoluble column 8 line 6, for "acetic" read acetate Signed and sealed this 8th day of October 1957.

(SEAL) Attest:

KARL H. AXLI NE ROBERT C. WATSON Attesting Oi'ficr Comnissioner of Patents 

2. A DIAGNOSTIC COMPOSITION IN TABLET FORM COMPRISING A MEMBER OF THE GROUP CONSISTING OF O-TOLIDINE, O-TOLUIOXIDE WHICH WILL LIBERATE HYDROGEN PEROXIDE, WATER DINE, BENZIDINE AND O-TOLUIDINE DIHYDROCHLORIDE, A PERSOLUBLE SOLID ACID STRONGER THAN ACETIC ACID AN ACETATE SALT WHICH WILL REACT WITH THE LAST SAID ACID TO PRODUCE ACETIC, ACID, AN EFFERVESCENT COUPLE, AND A WATER-SOLUBLE MATERIAL CONTRASTING IN COLOR WITH THE COLOR FORMED IN COMBINATION WITH BLOOD BY THE SELECTED MEMBER OF SAID GROUP. 